This R21 proposal is a collaborative effort of Dr. Liu (University of North Carolina) and Dr. Park (Children's Hospital/Harvard Medical School) to investigate the role of heparan sulfate (HS) modifications in inhibiting Pseudomonas aeruginosa lung infection. HS is a highly sulfated polysaccharide present in large amounts on the cell surface and in the extracellular matrix. HS serves as an important regulatory factor in a wide range of biological processes, including cell adhesion, migration and proliferation, blood coagulation, and microbial infections. The overall hypothesis that will be tested in this proposal is that uniquely sulfated HSPG sequences differentially modulate bacterial pathogenesis and host defense mechanisms. Our preliminary studies demonstrated that a unique 3-O-sulfated HS, biosynthesized by the HS 3-O-sulfotransferase isoform 3 (3OST- 3), significantly inhibits P. aeruginosa lung infection in mice. Specific Aim 1 will determine the biological basis of how 3-O-sulfated HS inhibits P. aeruginosa lung pathogenesis using a glycomics approach. We will determine the effects of 3-O-sulfation on the capacity of HS polysaccharides to regulate soluble antimicrobial factors, CXC chemokine-induced neutrophil recruitment, and anti-bacterial mechanisms of neutrophils. We will also determine if the expression of 3OST-3 is induced during P. aeruginosa infection as a host defense mechanism. Specific Aim 2 will define the minimum structural features of HS oligosaccharides that inhibit P. aeruginosa lung infection, and determine if HS modification is a target for anti-P. aeruginosa pneumonia therapy. We will identify the 3-O-sulfated HS sequences that are most effective in inhibiting P. aeruginosa lung infection. Successful completion of these studies should provide a mechanistic foundation for the development of novel HS-based therapies against P. aeruginosa pneumonia. PUBLIC HEALTH RELEVANCE: Pathogens establish the infection by exploiting heparan sulfate, a highly sulfated polysaccharide, present on the cell surface and extracellular matrix. This application will investigate the structural specificity of heparan sulfate for inhibiting infections caused by Pseudomonas aeruginosa. The results from study could lead to a better understanding on the bacterial infection mechanism and develop heparan-based antibacterial agents.